The objective of this proposal is to characterize the processing mechanisms of messenger RNA synthesized by adenovirus type 2 during productive infection. These studies will include the analysis of viral RNA synthesized in vivo and in vitro. In particular, the propoed research will focus on RNA transcribed from early region 2 (E2) which specifies a 72K DNA-binding protein involved in DNA synthesis and regulation of transcription of early viral RNAs. We have developed an in vitro system that utilizes whole cell extracts which is able to splice purified #2 RNAs, excising sequences from two introns and attaching leader sequences to the mRNA body. This system will be utilized to characterize the biomedical properties of the splicing mechanism and further purify the splicing enzymes. In particular, we will determine the nucleotide sequence of the splice junctions for the E2 in vitro processed RNAs. These experiments will determine the fidelity of the in vitro splicing reaction and also the accuracy of the in vitro splicing after each purification step of the whole cell extract. We will also analyze the requirements as well as the role of small nuclear RNAs in in vitro splicing. We will also construct unique viral DNA fragments cloned in the DNA single-stranded bacteriophage M-13, specific for introns, exons and leader sequences of the E2 mRNA. These probes will facilitate and simplify the assay of the E2 in vitro processed RNAs. We will attempt by biochemical standard techniques to further purify the splicing enzymes. We willalso analyze coupled in vitro transcription and splicing of viral RNAs. In addition, mRNA processing and mechanisms involved in the regulation of synthesis of individual mRNA families will be studied in vivo.